Method for typing and detecting HBV

ABSTRACT

The present invention relates to a method for detection and/or genetic analysis of HBV in a biological sample, comprising hybridizing the polynucleic acids of the sample with a combination of at least two nucleotide probes, with said combination hybridizing specifically to a mutant target sequence chosen from the HBV RT pol gene region and/or to a mutant target sequence chosen from the HBV preCore region and/or to a mutant target sequence chosen from the HBsAg region of HBV and/or to a HBV genotype-specific target sequence, with said target sequences being chosen from FIG.  1 , and with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to the polynucleic acids of the sample under the same hybridization and wash conditions, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence where T of said target sequence is replaced by U; and detecting the hybrids formed; and inferring the HBV genotype and/or mutants present in said sample from the differential hybridization signal(s) obtained. The invention further relates to sets of nucleotide probes and possibly primers useful in said methods as well as to their use in a method for typing and/or detecting HBV and to assay kits using the same.

The present application is a divisional of U.S. application Ser. No.10/453,792, filed Jun. 4, 2003, now U.S. Pat. No. 7,313,357, issued Dec.25, 2007which is a divisional of U.S. application Ser. No. 09/155,885,filed Oct. 8, 1998, now U.S. Pat. No. 6,709,812, issued Mar. 23, 2004,which is a 371 of U.S. National Phase of International Application No.PCT/EP97/02002, filed Apr. 21, 1997, which designated the U.S. andclaims benefit of EP 96870053.4, filed Apr. 19, 1996, the entirecontents of each of which is hereby incorporated by reference in thisapplication.

The present invention relates to the field of Hepatitis B virus (HBV)diagnosis. More particularly, the present invention relates to the fieldof HBV genotyping and/or determination of the presence of HBV mutants intest samples.

The present invention relates particularly to a method for the rapid andreliable detection of HBV mutants and/or genotypes occuring in a testsample using specific sets of probes optimized to function together in areverse-hybridisation assay.

Hepatitis B virus is a small enveloped DNA virus of approximately 3200bp long. Historically it has been characterized on the basis ofimmunological reaction of the HBsAg with sets of monoclonal antibodies.Isolates were described as a, indicating the common determinant for alldifferent subtypes, followed by subtype-specific combinations: dw, dr,yw, or yr. The latter are mutually exclusive pairs of determinants,covering the HBsAg amino acids 122 (d=lys, y=arg) and 160 (w=lys,r=arg). Several subdeterminants for w exist and can be ascribed to theappearance of certain amino acid variants at codon 127. More recently, agenetic classification has been proposed, based on molecular analysis ofthe virus. This kind of analysis showed that in total six differentgenotypes exist, indicated from A to F, with a maximum geneticdivergence of 8% when comparing complete genomes (reviewed by Magniusand Norder, 1995).

The genetic variability of HBV might be clinically important. Indeed,the genome variability might include some mechanisms by which HBV avoidsimmune clearance, and hence induces chronic infection. An importantprotein marker in inducing immune tolerance, virus elimination, andchronic infection, is HBeAg. The expression of this protein is strictlycontrolled both at the transcriptional and translational level (Li etal., 1993; Okamoto et al., 1990; Yuan et al., 1995; Sato et al., 1995).Therefore, in the natural course of HBV infection, a well characterizedstage of the disease is indicated as HBe-negative chronic hepatitis B(reviewed by Hadziyannis S. J., 1995). This phase is mostly due to theappearance of preCore translational stop codon mutations. The overalgenetic variability determines the frequency and physical location onthe viral genome where these translational stop-codon mutations appear.The transcriptional regulation was proposed to be the mechanism forgenotype A (and possibly also F), whereas the translational control wasmore likely to be found in the other genotypes (Li et al.; 1993; Sato etal., 1995). Contradictory to the translational regulation, it was shownthat the transcriptional regulation was unable to block the HBeAgexpression completely and was therefore proposed to categorize thephenotype of this mutant as HBe-suppressed, rather than as HBe-negative(Takahashi et al., 1995). In any case, these preCore mutants would leadto a destruction of the pre-existing balance between HBeAg incirculation and the HBc-derived peptides presented by class I HLAmolecules on the surface of infected hepatocytes, thereby diminishingthe supressive effect of HBeAg on T cells, finally resulting in partialliberation of core-specific CTLs and leading to apoptosis of theinfected hepatocytes. In general, after the emergence of the HBe-minusvariants, the course of the viral infection is characterized by theprogression of chronic hepatitis, which may lead to the development ofcirrhosis and hepatocellular carcinoma (Hadziyannis, 1995).

Another issue for which the genetic variability or genotyping of thevirus might be of relevance is in the development of vaccines where theresponse may be mediated by the virus type. Protection against HBVinfection of all subtypes is conferred by antibodies to the common ‘a’determinant of the HB surface antigen (HBsAg). It has been shown thatthis ‘a’ determinant presents a number of epitopes, and that itstertiary structure is most important for its antigenicity. The mostimportant region lies between amino acid 124 and 147, but can beextended from amino acid 114 to 150. An adequate anti-HBs response,built up after vaccination, is in principle fully protective. Infectionwith a HBV strain harboring mutations in the ‘a’ determinant regionmight result in vaccine failure, because the vaccine-induced humoralimmune response does not recognize the mutant HBsAg. The most commonvaccine-associated escape mutants are the substitutions of a glycine atposition 145 to an arginine (G145R), K141E, and T126N. But a 2-aainsertion between aa position 122 and 123, and 8-aa insertion between aa123 and 124 have also been found (Carman et al., 1990, 1995; Crawford,1990; Waters et al., 1992).

Lamivudine is a (−) enantiomer of 3′ thiacytidine, a2′3′-dideoxynucleoside analogue, and is known to be a potent inhibitorof HBV replication through inhibition of the reverse transcriptase (RT)activity of the HBV polymerase. Lamivudine treatment can result inhistological improvements in chronic hepatitis patients, and when givenpre- and post-liver transplantation, it can prevent graft reinfection(Honkoop et al., 1995; Naoumov et al., 1995). However, after treatment,a hepatitis flare-up can be observed in most patients, with ALTelevations and HBV DNA that becomes detectable again. This HBV DNArebound is associated with a new quasi species equilibrium, in a fewcases, virus breakthrough during therapy was observed, due to theselection of lamivudine resistent HBV strains. The exact nature of thisbreakthrough has been ascribed to the accumulation of mutations in theRT part of the Polymerase. A similar mechanism in the HIV RT polymerasehas been found, where upon lamivudine treatment, mutations accumulate inthe YMDD motif (Gao et al., 1993). This YMDD motif is also present inthe RT part of the HBV polymerase, and lamivudine-selected mutations inHBV have been found in this region (Tipples et al., 1996), as well as inother regions of the RT part of the polymerase (Ling et al., 1996).Penciclovir is another drug that has been shown to inhibit the reversetranscriptase activity of the HBV polymerase (Shaw et al., 1996), andmutations in the HBV polymerase may also be detected upon treatment withthis drug.

From all this it can be concluded that the information on the followingissues is essential for proper in vitro diagnosis, monitoring andfollow-up of HBV infections:

-   -   genotype;    -   preCore mutations;    -   vaccine escape mutations;    -   RT gene mutations selected by treatment with drugs such as        lamivudune and penciclovir.        To obtain all this information using existing technologies is        complicated, time-consuming, and requires highly-skilled and        experienced personel.

It is thus an aim of the present invention to develop a rapid andreliable detection method for determination of the presence or absenceof one or more HBV genotypes possibly present in a biological sample.

More particularly, it is an aim of the present invention to develop arapid and reliable detection method for determination of the presence orabsence of one or more variations in the HBV preS1 region and/or in theHBsAg region representing one or more HBV genotypes possibly present ina biological sample in one single experiment.

More particularly, it is an aim of the present invention to develop arapid and reliable detection method for determination of the presence orabsence of one or more HBV mutants possibly present in a biologicalsample in one single experiment.

More particularly, ft is an aim of the present invention to develop arapid and reliable detection method for determination of one or moremutations in the preCore region of HBV possibly present in a biologicalsample in one single experiment.

More particularly, it is an aim of the present invention to develop arapid and reliable detection method for determination of one or moremutations in the HBsAg region of HBV possibly present in a biologicalsample in one single experiment.

More particularly, it is an aim of the present invention to develop arapid and reliable detection method for determination of one or moremutations in the polymerase (pol) gene region of HBV possibly present ina biological sample in one single experiment.

More particularly, it is an aim of the present invention to develop arapid and reliable detection method for the simultaneous determinationof one or several HBV genotypes in combination with one or several HBVmutants possibly present in a biological sample in one singleexperiment.

It is also an aim of the present invention to provide a genotyping assayor method which allows to infer the nucleotide sequence at codons ofinterest and/or the HBV mutants of interest, and/or infer the HBVgenotype possibly present in a biological sample.

Even more particularly it is also an aim of the present invention toprovide a genotyping assay allowing the detection of the different HBVmutants and genotypes in one single experimental setup.

It is another aim of the present invention to select particular probesable to discriminate one or more HBV mutations in one of the abovementioned regions of the HBV genome and/or able to discriminate one ormore HBV genotypes.

It is more particularly an aim of the present invention to selectparticular probes able to discriminate wild-type HBV from mutant HBVsequences.

It is also an aim of the present invention to select particular probesable to discriminate wild-type and polymorphic variants of HBV frommutant HBV sequences.

It is also an aim of the present invention to select particular probesable to discriminate HBV genotype sequences.

It is moreover an aim of the present invention to combine a set ofselected probes able to genotype HBV and/or discriminate different HBVmutants possibly present in a biological sample, whereby all probes canbe used under the same hybridisation and wash conditions.

It is also an aim of the present invention to select primers enablingthe amplification of the gene fragment(s) determining the HBV genomicmutations or variations of interest as discussed above.

The present invention also aims at diagnostic kits comprising saidprobes useful for developing such a genotyping assay and/or assays fordetecting, monitoring or following-up HBV infection and/or assays fordetecting HBV mutations.

All the aims of the present invention have been met by the followingspecific embodiments.

As a solution to the above-mentioned problem that it is essential forproper diagnosis, monitoring and follow-up of HBV infection to haveinformation on the genotype of HBV present, the present inventionprovides an elegant way to tackle problems of such complexity whichinvolves residing to a reverse hybridization approach (particularly onLine Probe Assays strips, as described by Stuyver et al., 1993). Usingthis technology it is possible to conveniently obtain all essential datain one test run. To achieve this goal, a set of probes needs to bedesigned and assembled which can detect all relevant polymorphisms inthe HBV gene regions of interest.

The present invention thus particularly relates to a method fordetermining the presence or absence of one or more HBV genotypes in abiological sample, comprising:

-   (i) if need be releasing, isolating or concentrating the polynucleic    acids present in the sample;-   (ii) if need be amplifying the relevant part of a suitable HBV gene    present in said sample with at least one suitable primer pair;-   (iii) hybridizing the polynucleic acids of step (i) or (ii) with at    least two nucleotide probes hybridizing specifically to a HBV    genotype specific target sequence chosen from FIG. 1; with said    probes being applied to known locations on a solid support and with    said probes being capable of hybridizing to polynucleic acids of    step (i) or (ii) under the same hybridization and wash conditions or    with said probes hybridizing specifically with a sequence    complementary to any of said target sequences, or a sequence wherein    T of said target sequence is replaced by U;-   (iv) detecting the hybrids formed in step (iii);-   (v) inferring the HBV genotype present in said sample from the    differential hybridization signal(s) obtained in step (iv).

The genotype specific target sequences can be any nucleotide variationappearing upon alignment of the different HBV genomes that permitsclassification of a certain HBV isolate as a certain genotype (see FIG.1).

The expression “relevant part of a suitable HBV gene” refers to the partof the HBV gene encompassing the HBV genotype specific target sequencechosen from FIG. 1 to be detected.

According to a preferred embodiment of the present invention, step (iii)is performed using a set of at least 2, preferably at least 3, morepreferably at least 4 and most preferably at least 5 probes allmeticulously designed such that they show the desired hybridizationresults, when used in a reverse hybridisation assay format, moreparticularly under the same hybridization and wash conditions implyingthat each of said probes is able to form a complex upon hybridisationwith its target sequence present in the polynucleic acids of the sampleas obtained after step (i) or (ii).

The numbering of the HBV gene encoded amino acids and nucleotides is asgenerally accepted in literature.

More particularly, the present invention relates to a set of at least 2probes allowing the detection of a genotype specific variation, possiblyalso including one or more probes allowing the detection of a wild-typesequence, a polymorphic or a mutated sequence at any one of thenucleotide positions showing a sequence diversity upon alignment of allknown or yet to be discovered HBV sequences as represented in FIG. 1 forall complete HBV genomes found in the EMBL/NCBI/DDBJ/Genbank.

The sets of probes according to the present invention have as a commoncharacteristic that all the probes in said set are designed so that theycan be used together in a reverse-hybridization assay, more particularlyunder similar or identical hybridization and wash conditions asindicated above and below.

Selected sets of probes according to the present invention includeprobes which allow to differentiate any of the HBV genotype specificnucleotide changes as represented in FIG. 1, preferably in the preS1 orHBsAg region of HBV. Said probes being characterized in that they canfunction in a method as set out above.

In order to solve the above-mentioned problem of obtaining informationon the possible presence of HBV mutants in a given sample, the presentinvention provides an elegant way to tackle this problem which involvesresiding to a reverse hybridisation approach (particularly on Line ProbeAssays strips, as described by Stuyver et al., 1993). Using thistechnology it is possible to conveniently obtain all essential data inone test run. To achieve this goal, a set of probes needs to be designedand assembled which can detect all relevant mutations and possibly alsowild-type sequences or polymorphisms in the HBV gene regions ofinterest.

Another particularly preferred embodiment of the present invention thusis a method for determining the presence or absence of one or more HBVmutants in a biological sample, comprising:

-   (i) if need be releasing, isolating or concentrating the polynucleic    acids present in the sample;-   (ii) if need be amplifying the relevant part of a suitable HBV gene    present in said sample with at least one suitable primer pair;-   (iii) hybridizing the polynucleic acids of step (i) or (ii) with at    least two nucleotide probes hybridizing specifically to a HBV mutant    target sequence chosen from FIG. 1, with said probes being applied    to known locations on a solid support and with said probes being    capable of hybridizing to the polynucleic acids of step (i) or (ii)    under the same hybridization and wash conditions, or with said    probes hybridizing specifically with a sequence complementary to any    of said target sequences, or a sequence wherein T of said target    sequence is replaced by U and with said set or probes possibly also    comprising one or more wild-type HBV probes corresponding with the    respective mutated HBV target sequence;-   (iv) detecting the hybrids formed in step (iii);-   (v) inferring the HBV mutant(s) present in said sample from the    differential hybridization signal(s) obtained in step (iv).

It is to be understood that the term “mutant target sequence” not onlycovers the sequence containing a mutation, but also the correspondingwild-type sequence. The HBV mutant target sequence according to thepresent invention can be any sequence including a HBV mutated codonknown in the art or yet to be discovered. Particularly preferred HBVmutant target regions are set out below.

In order to solve the problem as referred to above of obtaininginformation on the essential issues for proper diagnosis of HBV (namelygenotype and different mutations particularly mutations in the preCoreregion, vaccine escape mutations and RT gene mutations selected bytreatment with drugs such as lamivudine and penciclovir), the presentinvention provides a particularly elegant way to obtain such complexinformation.

Moreover, careful analysis of the data obtained by the present inventorsclearly revealed that combining the information concerning the preCoreand escape mutants with data on the genotype is essential to allowadequate interpretation of the results. Hence it is highly advantageousto be able to produce all relevant data simultaneously.

In this method for diagnosing HBV mutants, preferably in combinationwith HBV genotyping, a set of probes selected as defined above may beused, wherein said set of probes is characterized as being chosen suchthat for a given HBV mutation, the following probes are included in saidset:

-   -   at least one probe for detecting the presence of the mutated        nucleotide(s) at said position;    -   at least one probe for detecting the presence of the wild-type        nucleotide(s) at said position;    -   possibly also (an) additional probe(s) for detecting wild-type        polymorphisms at positions surrounding the mutation position.        Inclusion of the latter two types of probes greatly contributes        to increasing the sensitivity of said assays as demonstrated in        the examples section.

Selected sets of probes according to the present invention include atleast one probe, preferably at least two probes, characterizing thepresence of a HBV mutation at nucleotide positions chosen from thepreCore region of HBV, more particularly from the following list ofcodons susceptible to mutations in the HBV preCore region, such as codon15 in genotype A, and for all genotypes: codon 28, codon 29, and codon28 and 29, or in the preCore promoter region (see FIG. 1).

Said probes being characterized in that they can function in a method asset out above.

An additional embodiment of the present invention includes at least oneprobe, preferably at least two probes, characterizing the presence of avaccine escape mutation in codon positions chosen from the HBsAg regionof HBV, more particularly from the list of codons susceptible tomutations in the HBV HBsAg region, such as at codons 122, 126, 141, 143,144 or 145 (see FIG. 1).

An additional embodiment of the present invention includes at least oneprobe, preferably at least two probes, characterizing the presence of amutation in the RT pol gene region of HBV, that gives rise to resistanceto drugs such as lamivudine and penciclovir, for instance mutation of Mto V or to I at position 552 (in the YMDD motif), mutation of V to I atposition 555, mutation of F to L at position 514, mutation of V to L atposition 521, mutation of P to L at position 525 and mutation of L to Mat position 528 (see FIG. 1).

In a selected embodiment, a combination of at least two oligonucleotideprobes is used and said combination of probes hybridizes specifically toat least two of the following groups of target sequences:

a mutant target sequence chosen from the HBV RT pol gene region,

a mutant target sequence chosen from the HBV preCore region,

a mutant target sequence chosen from the HBsAg region of HBV,

a HBV genotype-specific target sequence.

For instance, an embodiment involves hybridizing with at least onenucleotide probe hybridizing specifically to a genotype specific targetsequence chosen from FIG. 1 and at least one nucleotide probehybridizing specifically to a HBV mutant target sequence chosen fromFIG. 1.

Another selected embodiment involves, for instance, hybridizing with atleast one nucleotide probe hybridizing specifically to a genotypespecific target sequence chosen from FIG. 1 and at least one nucleotideprobe hybridizing specifically to a HBV mutant target sequence chosenfrom the RT pol gene region as represented in FIG. 1.

Another selected embodiment involves, for instance, hybridizing with atleast one nucleotide probe hybridizing specifically to a genotypespecific target sequence chosen from FIG. 1 and at least one nucleotideprobe hybridizing specifically to a HBV mutant target sequence chosenfrom the preCore region as represented in FIG. 1.

Another selected embodiment involves, for instance, hybridizing with atleast one nucleotide probe hybridizing specifically to a genotypespecific target sequence chosen from FIG. 1 and at least one nucleotideprobe hybridizing specifically to a HBV vaccine escape mutant targetsequence within the HBsAg region as represented in FIG. 1.

In a selected embodiment, a combination of at least threeoligonucleotide probes is used and said combination of probes hybridizesspecifically to at least three of the following groups of targetsequences:

a mutant target sequence chosen from the HBV RT pol gene region,

a mutant target sequence chosen from the HBV preCore region,

a mutant target sequence chosen from the HBsAg region of HBV,

a HBV genotype-specific target sequence.

For instance, an embodiment involves hybridizing with at least onenucleotide probe hybridizing specifically to a genotype specific targetsequence chosen from FIG. 1, and at least one nucleotide probehybridizing specifically to a HBV mutant target sequence chosen from thepreCore region as represented in FIG. 1, and at least one nucleotideprobe hybridizing specifically to a HBV vaccine escape mutant targetsequence chosen from the HBsAg region as represented in FIG. 1.

For instance, another embodiment involves hybridizing with at least oneprobe hybridizing specifically to a mutant target sequence from the HBVRT-pol gene region of HBV, and at least one probe hybridizingspecifically to a mutant target sequence from the HBsAg region of HBV,and at least one probe hybridizing specifically to a genotype-specifictarget sequence from the HBsAg region of HBV. According to thisembodiment, the relevant part of the HBV genome can be amplified by useof one primer pair, for instance HBPr 75 and HBPr 94.

In a selected embodiment, a combination of at least four oligonucleotideprobes is used and said combination of probes hybridizes specifically toall of the following groups of target sequences:

a mutant target sequence chosen from the HBV RT pol gene region,

a mutant target sequence chosen from the HBV preCore region,

a mutant target sequence chosen from the HBsAg region of HBV,

a HBV genotype-specific target sequence.

Particularly preferred embodiments of the invention thus include a setof probes as set out above comprising at least one, preferably at leasttwo, at least three, at least four or more probe(s) for targeting one,preferably two, three or more nucleotide changes appearing in thealignment of HBV genomes as represented in FIG. 1.

Even more, preferred selected sets of probes according to the presentinvention include probes derived from two of the same or differentregions of HBV bearing HBV mutated nucleotides, or in addition also athird (set of) probe(s) characterizing the presence of a third HBVmutation at any of the positions shown in FIG. 1, or particularcombinations thereof.

Particularly preferred is also a set of probes which allows simultaneousdetection of HBV mutations at codons 15, 28 and 29 in the preCoreregion, possibly in combination with mutations in the preCore promoterregions, in combination with mutations at codons 122, 126, 141, 143,144, 145 in the HBsAg region, possibly also in combination withmutations in the HBV pol gene at codons 514, 521, 525, 528, 552 or 555.

In the instances where the alignment of HBV genomes of FIG. 1 isreferred to in this invention, it should be construed as referring to analignment of all existing and future HBV genomes. The existing HBVgenome sequences can be deduced from any database, such as theEMBL/NCBI/DDBJ/GENBANK database.

A preferred set of preCore, preS1, HBsAg and RT pol gene probes of thisinvention are the probes with SEQ ID NO 1 to 278 of Table 1 (see alsoFIG. 1).

Particularly preferred sets of probes in this respect are shown in FIG.2 and in FIG. 4. The probes in FIG. 2 and in FIG. 4 were withheld aftera first selection for preCore, preS1, HBsAg and RT pol probes.

The probes of the invention are designed for obtaining optimalperformance under the same hybridization conditions so that they can beused in sets of at least 2 probes for simultaneous hybridization. Thishighly increases the usefulness of these probes and results in asignificant gain in time and labour. Evidently, when other hybridizationconditions would be preferred, all probes should be adapted accordinglyby adding or deleting a number of nucleotides at their extremities. Itshould be understood that these concomitant adaptations should give riseto essentially the same result, namely that the respective probes stillhybridize specifically with the defined target. Such adaptations mightalso be necessary if the amplified material should be RNA in nature andnot DNA as in the case for the NASBA system.

The selection of the preferred probes of the present invention is basedon a reverse hybridization assay format using immobilizedoligonucleotide probes present at distinct locations on a solid support.More particularly the selection of preferred probes of the presentinvention is based on the use of the Line Probe Assay (LiPA) principlewhich is a reverse hybridization assay using oligonucleotide probesimmobilized as parallel lines on a solid support strip (Stuyver et al.1993; international application WO 94/12670). This approach isparticularly advantageous since it is fast and simple to perform. Thereverse hybridization format and more particularly the LiPA approach hasmany practical advantages as compared to other DNA techniques orhybridization formats, especially when the use of a combination ofprobes is preferable or unavoidable to obtain the relevant informationsought.

It is to be understood, however, that any other type of hybridizationassay or format using any of the selected probes as described further inthe invention, is also covered by the present invention.

The reverse hybridization approach implies that the probes areimmobilized to certain locations on a solid support and that the targetDNA is labelled in order to enable the detection of the hybrids formed.

The following definitions serve to illustrate the terms and expressionsused in the present invention.

The term “genetic analysis” refers to the study of thenucleotide_sequence of the genome of HBV by any appropriate technique.

The term “HBV mutant” refers to any HBV strain harbouring genomicvariations with serological, genetical or clinical consequences.

The term “vaccine escape mutant” is reviewed in the introduction sectionand in Example 7. The most important region lies between amino acid 124and 147of the HBsAg region, but can be extended from amino acid 114 to150.

The term “mutant resistant to drugs such as lamivudine and penciclovir”is reviewed in the introduction section and in Example 8.

The term “HBV genotype” refers to HBV strains with an intergenotypevariation of 8% or more based on a comparison of complete genomes.

The target material in the samples to be analyzed may either be DNA orRNA, e.g. genomic DNA, messenger RNA, viral RNA or amplified versionsthereof. These molecules are also termed polynucleic acids.

It is possible to use genomic DNA or RNA molecules from samplessusceptible of containing HBV in the methods according to the presentinvention.

Well-known extraction and purification procedures are available for theisolation of RNA or DNA from a sample (f.i. in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarbourLaboratory Press (1989)).

The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected.

The term “target sequence” as referred to in the present inventiondescribes the nucleotide sequence of a part of wild-type, polymorphic ormutant HBV gene sequence to be specifically detected by a probeaccording to the present invention. The polymorphic sequence mayencompass one or more polymorphic nucleotides; the mutant sequence mayencompass one or more nucleotides that are different from the wild-typesequence. It is to be understood that the term “mutant target sequence”not only covers the sequence containing a mutation, but also thecorresponding wild-type sequence. Target sequences may generally referto single nucleotide positions, codon positions, nucleotides encodingamino acids or to sequences spanning any of the foregoing positions, inthe present invention said target sequence often includes one, two ormore variable nucleotide positions, in the present invention polynucleicacids detected by the probes of the invention will comprise the targetsequence against which the probe is detected.

It is to be understood that the complement of said target sequence isalso a suitable target sequence in some cases. The target sequences asdefined in the present invention provide sequences which should at leastbe complementary to the central part of the probe which is designed tohybridize specifically to said target region. In most cases the targetsequence is completely complementary to the sequence of the probe.

The term “complementary” as used herein means that the sequence of thesingle stranded probe is exactly the (inverse) complement of thesequence of the single-stranded target, with the target being furtherdefined as the sequence where the mutation to be detected is located.

Since the current application requires the detection of single basepairmismatches, stringent conditions for hybridization are required,allowing in principle only hybridization of exactly complementarysequences. However, variations are possible in the length of the probes(see below), it should also be noted that, since the central part of theprobe is essential for its hybridization characteristics, possibledeviations of the probe sequence versus the target sequence may beallowable towards head and tail of the probe when longer probe sequencesare used. These variations, which may be conceived from the commonknowledge in the art, should however always be evaluated experimentally,in order to check if they result in equivalent hybridizationcharacteristics as the exactly complementary probes.

Preferably, the probes of the invention are about 5 to 50 nucleotideslong, more preferably from about 10 to 25 nucleotides. Particularlypreferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in thepresent invention may be ribonucleotides, deoxyribonucleotides andmodified nucleotides such as inosine or nucleotides containing modifiedgroups which do not essentially alter their hybridisationcharacteristics.

Probe sequences are represented throughout the specification as singlestranded DNA oligonucleotides from the 5′ to the 3′ end. It is obviousto the man skilled in the art that any of the below-specified probes canbe used as such, or in their complementary form, or in their RNA form(wherein T is replaced by U).

The probes according to the invention can be prepared by cloning ofrecombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead) or a chip. Prior to application to the membrane orfixation it may be convenient to modify the nucleic acid probe in orderto facilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

The term “labelled” refers to the use of labelled nucleic acids.Labelling may be carried out by the use of labelled nucleotidesincorporated during the polymerase step of the amplification such asillustrated by Saiki et al. (1988) or Bej et al. (1990) or labelledprimers, or by any other method known to the person skilled in the art.The nature of the label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic(biotin, digoxigenin, etc.).

The term “primer” refers to a single stranded oligonucleotide sequencecapable of acting as a point of initiation for synthesis of a primerextension product which is complementary to the nucleic acid strand tobe copied. The length and the sequence of the primer must be such thatthey allow to prime the synthesis of the extension products. Preferablythe primer is about 5-50 nucleotides long. Specific length and sequencewill depend on the complexity of the required DNA or RNA targets, aswell as on the conditions of primer use such as temperature and ionicstrength.

The expression “suitable primer pair” in this invention refers to a pairof primers allowing the amplification of part or all of the HBV gene forwhich probes are immobilized.

The fact that amplification primers do not have to match exactly withthe corresponding template sequence to warrant proper amplification isamply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of Qβ replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules known in the art.

The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the originalDNA sequences of the invention these variations will necessitateadaptions with respect to the conditions under which the oligonucleotideshould be used to obtain the required specificity and sensitivity.However the eventual results of hybridisation will be essentially thesame as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of theoligonucleotide molecules, etc.

The “sample” may be any biological material taken either directly fromthe infected human being (or animal), or after culturing (enrichment).Biological material may be e.g. expectorations of any kind,broncheolavages, blood, skin tissue, biopsies, sperm, lymphocyte bloodculture material, colonies, liquid cultures, faecal samples, urine etc.

The sets of probes of the present invention will include at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may beapplied in two or more (possibly as many as there are probes) distinctand known positions on a solid substrate. Often it is preferable toapply two or more probes together in one and the same position of saidsolid support.

For designing probes with desired characteristics, the following usefulguidelines known to the person skilled in the art can be applied.

Because the extent and specificity of hybridization reactions such asthose described herein are affected by a number of factors, manipulationof one or more of those factors will determine the exact sensitivity andspecificity of a particular probe, whether perfectly complementary toits target or not. The importance and effect of various assayconditions, explained further herein, are known to those skilled in theart.

The stability of the [probe:target] nucleic acid hybrid should be chosento be compatible with the assay conditions. This may be accomplished byavoiding long AT-rich sequences, by terminating the hybrids with G:Cbase pairs, and by designing the probe with an appropriate Tm. Thebeginning and end points of the probe should be chosen so that thelength and % GC result in a Tm about 2-10° C. higher than thetemperature at which the final assay will be performed. The basecomposition of the probe is significant because G-C base pairs exhibitgreater thermal stability as compared to A-T base pairs due toadditional hydrogen bonding. Thus, hybridization involving complementarynucleic acids of higher G-C content will be stable at highertemperatures.

Conditions such as ionic strength and incubation temperature under whicha probe will be used should also be taken into account when designing aprobe. It is known that hybridization will increase as the ionicstrength of the reaction mixture increases, and that the thermalstability of the hybrids will increase with increasing ionic strength.On the other hand, chemical reagents, such as formamide, urea, DMSO andalcohols, which disrupt hydrogen bonds, will increase the stringency ofhybridization. Destabilization of the hydrogen bonds by such reagentscan greatly reduce the Tm. In general, optimal hybridization forsynthetic oligonucleotide probes of about 10-50 bases in length occursapproximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum may allow mismatched basesequences to hybridize and can therefore result in reduced specificity.

It is desirable to have probes which hybridize only under conditions ofhigh stringency. Under high stringency conditions only highlycomplementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. The degree of stringency is chosen such as to maximize thedifference in stability between the hybrid formed with the target andthe nontarget nucleic acid. In the present case, single base pairchanges need to be detected, which requires conditions of very highstringency.

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, varying in locationand length, which will yield probes with the desired hybridizationcharacteristics. In other cases, one sequence may be significantlybetter than another which differs merely by a single base. While it ispossible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly complementary base sequencewill normally primarily determine hybrid stability. Whileoligonucleotide probes of different lengths and base composition may beused, preferred oligonucleotide probes of this invention are betweenabout 5 to 50 (more particularly 10-25) bases in length and have asufficient stretch in the sequence which is perfectly complementary tothe target nucleic acid sequence.

Regions in the target DNA or RNA which are known to form strong internalstructures inhibitory to hybridization are less preferred. Likewise,probes with extensive self-complementarity should be avoided. Asexplained above, hybridization is the association of two single strandsof complementary nucleic acids to form a hydrogen bonded double strand.It is implicit that if one of the two strands is wholly or partiallyinvolved in a hybrid that it will be less able to participate information of a new hybrid. There can be intramolecular andintermolecular hybrids formed within the molecules of one type of probeif there is sufficient self complementarity. Such structures can beavoided through careful probe design. By designing a probe so that asubstantial portion of the sequence of interest is single stranded, therate and extent of hybridization may be greatly increased. Computerprograms are available to search for this type of interaction. However,in certain instances, it may not be possible to avoid this type ofinteraction.

Standard hybridization and wash conditions are disclosed in theMaterials & Methods section of the Examples. Other conditions are forinstance 3× SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at50° C.

Other solutions (SSPE (Sodium saline phosphate EDTA), TMACl (Tetramethylammonium Chloride), etc.) and temperatures can also be used providedthat the specificity and sensitivity of the probes is maintained. Ifneed be, slight modifications of the probes in length or in sequencehave to be carried out to maintain the specificity and sensitivityrequired under the given circumstances.

In a more preferential embodiment, the above-mentioned polynucleic acidsfrom step (i) or (ii) are hybridized with at least two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of theabove-mentioned target region specific probes, preferably with 5 or 6probes, which, taken together, cover the “mutation region” of therelevant HBV gene.

The term “mutation region” means the region in the relevant HBV genesequence where at least one mutation encoding a HBV mutant is located ina preferred part of this mutation region is represented in FIG. 1.

Apart from mutation regions as defined above the HBV wild-type or mutantgenomes may also show polymorphic nucleotide variations at positionsother than those referred to as genotype specific or mutant specificvariated positions as shown in FIG. 1.

Since some mutations may be more frequently occurring than others, e.g.in certain geographic areas or in specific circumstances (e.g. ratherclosed communities) it may be appropriate to screen only for specificmutations, using a selected set of probes as indicated above. This wouldresult in a more simple test, which would cover the needs under certaincircumstances.

In order to detect HBV genotypes and/or HBV mutants with the selectedset of oligonucleotide probes, any hybridization method known in the artcan be used (conventional dot-blot, Southern blot, sandwich, etc.).

However, in order to obtain fast and easy results if a multitude ofprobes are involved, a reverse hybridization format may be mostconvenient.

In a preferred embodiment the selected set of probes are immobilized toa solid support in known distinct locations (dots, lines or otherfigures). In another preferred embodiment the selected set of probes areimmobilized to a membrane strip in a line fashion. Said probes may beimmobilized individually or as mixtures to delineated locations on thesolid support.

A specific and very user-friendly embodiment of the above-mentionedpreferential method is the LiPA method, where the above-mentioned set ofprobes is immobilized in parallel lines on a membrane, as furtherdescribed in the examples.

The invention also provides for a set of primers allowing amplificationof the region of the respective HBV gene to be detected by means ofprobes. Examples of such primers of the invention are given in Table 1and FIG. 1.

Primers may be labelled with a label of choice (e.g. biotine). Differentprimer-based target amplification systems may be used, and preferablyPCR-amplification, as set out in the examples. Single-round or nestedPCR may be used.

The invention also provides a kit for detection and/or genetic analysisof HBV genotypes and/or HBV mutants present in a biological samplecomprising the following components:

-   (i) when appropriate, a means for releasing, isolating or    concentrating the polynucleic acids present in said sample;-   (ii) when appropriate, at Least one suitable primer pair;-   (iii) at least two of the probes as defined above, possibly fixed to    a solid support;-   (iv) a hybridization buffer, or components necessary for producing    said buffer;-   (v) a wash solution, or components necessary for producing said    solution;-   (vi) when appropriate, a means for detecting the hybrids resulting    from the preceding hybridization.-   (vii) when appropriate, a means for attaching said probe to a known    location on solid support.

The term “hybridization buffer” means a buffer enabling a hybridizationreaction to occur between the probes and the polynucleic acids presentin the sample, or the amplified products, under the appropriatestringency conditions.

The term “wash solution” means a solution enabling washing of thehybrids formed under the appropriate stringency conditions.

As illustrated in the Examples section, a line probe assay (LiPA) wasdesigned for screening for HBV genotypes and/or HBV mutants. Theprinciple of the assay is based on reverse hybridization of an amplifiedpolynucleic acid fragment such as a biotinylated PCR fragment of the HBVgene onto short oligonucleotides. The latter hybrid can then, via abiotine-streptavidine coupling, be detected with a non-radioactivecolour developing system.

The following examples only serve to illustrate the present invention.These examples are in no way intended to limit the scope of the presentinvention.

FIGURE AND TABLE LEGENDS

FIG. 1: Alignment of 35 complete HBV genomes, isolates belonging togenotype A are: HBVXCPS, HBVADW, HVHEPB, S50225, HPBADWZCG; genotype B:HPBADW3, HPBADWZ, HPBADW1, HPBADW2; genotype C: HPBCGADR, HBVADRM,HPBADRA, HPBCG, HEHBVAYR, HBVADR, HBVADR4, HPBADR1C, HPBADRC, HBVPREX,HPBETNC, HHVBC, HHVCCHA; genotype D: HBVAYWMCG, HBVAYWC, HBVAYWCI,HBVAYWE, HBVDNA, HPBHBVAA, XXHEPAV, HBVORFS; genotype E: HHVBE4,HHVBBAS; and genotype F: HHBF, HHVBFFOU, HBVADW4A. To preservealignment, several gaps were created in the alignment and are indicatedwith /. Positions of start and end of the different HBV encoded genes isindicated: HBsAg: hepatitis B surface antigen (small surface antigen);HBx: hepatitis B X protein; HB Pol: hepatitis B polymerase protein,encoding a terminal protein, a spacer, a RT/DNA polymerase region, andan RNAse H activity; HBcAg: hepatitis B Core antigen; HBpreS1Ag:hepatitis B preS1 antigen (large surface antigen); HBpreS2Ag: hepatitisB preS2 antigen (middle surface antigen). The position of the PCRprimers is indicated with a large box over all 35 sequences. Thepolarity of the PCR primer can be deduced from the position of the nameabove these boxes: left=antisense primer; right=sense primer. LiPAprobes are indicated with small boxes, the numbers of the probes areindicated next to the probes or to the right of the alignment, andcorrespond to the probe numbers in Table 1.

FIG. 2: LiPA HBV design. The content of a HBV LiPA strip is detailed.For each iine number, the region on the viral genome is indicated,together with the genotype that is detected, the probe number thatcorresponds with the boxes from the alignment in FIG. 1, and thesequence of the probe.

FIG. 3: Combined result of genotype determination in the preS1 regionand preCore scanning on 24 samples. The interpretation of each sample isgiven under each strip. Probe reactivities on lines 3 to 14 are obtainedfrom the preS1 PCR fragment, probe reactivities on lines 15 to 27 aredue to the preCore PCR fragment. Genotypes are indicated from A to F.The interpretation for the preCore region is as follows: W=wild type;M=mutant; I=indeterminate, meaning that no reactivity is observed, whichis due to mutations that could not yet be detected with the selectedprobes; mix=mixture of wild type and mutant; interpretation of codon 15is only relevant for genotype A, the absence of reactivity on HBPr 45for genotypes B to F is of no use as is indicated with—(not applicable).Since the presence or absence of preCore mutations has effect on theserological HBeAg status, this is also indicated.

FIG. 4: Probes used in HBV LiPA. Probes were designed for genotyping inthe HBsAg region and for detection of drug resistance mutations in theYMDD motif (see also FIG. 5), as well as for detection of mutations inthe pre Core region (see also FIG. 6).

FIG. 5: Example of a LiPA assay combining HBV genotyping in the HBsAgregion and detection of drug resistance mutations in the YMDD motif.Genotypes are indicated from A to F. The design of the strip is shown tothe right, with the numbers of the probes corresponding to the numbersin Table 1 and in FIG. 4. The genotypes and mutant motifs to which eachprobe hybridizes are written to the outer right. The combination ofreactive probes allows the determination of a unique genotype.

FIG. 6: Example of the determination of preCore mutations by the LiPAtechnique. The design of the strip is shown to the right, with thenumbers of the probes corresponding to the numbers in Table 1. Themutant target sequences to which the probes hybridize are indicated tothe outer right. Motif M2 corresponds to a mutation in codon 28, M4corresponds to a mutation in codon 29. M2/M4 has mutations in both 28and 29.

FIG. 7: Detection of a mutation in the YMDD motif of HBV pol upontreatment with lamivudune. The graph shows a time course of the viralload during lamivudine treatment. To the right LiPA strips are shown,corresponding to assays at the beginning of the treatment (5/95), 10months of treatment (2/96) and 14 months of treatment (6/96). The assayshows that during treatment the YMDD motif mutates to YVDD.

Table 1: Overview of all primers and probes referred to in the Figureswith an indication of their respective SEQ ID NO and the region of theHBV genome they are designed for. Primers from the PreS1 region include1, 106, 2 (sense primers) and 4, 107 and 3 (antisense primers). Primersfrom the HBsAg region include 75 and 104 (sense primers) and 76, 94 and105 (antisense primers). Primers from the PreCore region include 5, 6,69, 70, 84, 86, 87 and 108 (sense primers) and 7, 8, 85 and 109(antisense primers). The remaining oligonucleotides are probes from thePreCore, PreS1, HBsAg and RT pol gene regions of HBV as indicated. TheYMDDV motif and its mutants consist of amino acids 551 to 555 of the RTpol protein; the sequence MGVGL and its mutant consist of amino acids519 to 523 of the RT pol protein; the sequence SPFLL and its mutants andgenotypic variants consist of amino acids 524 to 528 of the RT polprotein.

TABLE 1 HBV probe and primer design SEQ ID Name Sequence NO Region HBPr1GGGTCACCATATTCTTGGG 1 preS1 primer sense HPBr2 GAACAAGAGCTACAGCATGGG 2preS1 primer sense HBPr3 CCACTGCATGGCCTGAGGATG 3 preS1 primer anti-senseHBPr4 GTTCCT/GGAACTGGAGCCACCAG 4 preS1 primer anti-sense HBPr5TCTTTGTATTAGGAGGCTGTAG 5 preCore primer sense HBPr6GCTGTAGGCATAAATTGGTCTG 6 preCore primer sense HBPr7CTCCACAGT/AAGCTCCAAATTC 7 preCore primer anti-sense HBPr8GAAGGAAAGAAGTCAGAAGGC 8 preCore primer anti-sense HBPr9 TGGCTTTGGGGCATGG9 preCore HBPr10 TGGCTTTAGGGCATGG 10 preCore HBPr11 TGGCTTTAGGACATGG 11preCore HBPr12 AAGTTGCATGGTGCTG 12 preCore HBPr13 CACCTCTGCCTAATCAT 13preCore HBPr14 TGGGGTGGAGCCCTCAG 14 preS1 HBPr15 GCCAGCAGCCAACCAG 15preS1 HBPr16 CCCATGGGGGACTGT 16 preS1 HBPr17 AACCCCAACAAGGATG 17 preS1HBPr18 TCCACCAGCAATCCT 18 preS1 HBPr19 TGGGGGAAGAATATTT 19 preS1 HBPr20AAATTCCAGCAGTCCC 20 preS1 HBPr21 GTTCCCAACCCTCTGG 21 preS1 HBPr22AACCTCGCAAAGGCAT 22 preS1 HBPr23 TGCATTCAAAGCCAAC 23 preS1 HBPr24TACTCACAACTGTGCC 24 preS1 HBPr25 ACCCTGCGTTCGGAGC 25 preS1 HBPr26CAGGAAGACAGCCTAC 26 preS1 HBPr27 GATCCAGCCTTCAGAG 27 preS1 HBPr28ATGCTCCAGCTCCTAC 28 preS1 HBPr29 GCTTTCTTGGACGGTC 29 preS1 HBPr30CTACCCCAATCACTCC 30 preS1 HBPr31 AGCACCTCTCTCAACG 31 preS1 HBPr32CCAATGGCAAACAAGG 32 preS1 HBPr33 CTGAGGGCTCCACCCCA 33 preS1 HBPr34ATGCAACTTTTTCACC 34 preCore HBPr35 ATCTCTTGTACATGTC 35 preCore HBPr36ATCTCATGTTCATGTC 36 preCore HBPr37 CAGTGGGACATGTACA 37 preCore HBPr38CAGTAGGACATGAACA 38 preCore HBPr39 CTGTTCAAGCCTCCAA 39 preCore HBPr40AGCCTCCAAGCTGTGC 40 preCore HBPr41 AAAGCCACCCAAGGCA 41 preCore HBPr42TGGCTTTAGGACATGGA 42 preCore HBPr43 GACATGTACAAGAGATGA 43 preCore HBPr44GACATGAACATGAGATGA 44 preCore HBPr45 TGTACATGTCCCACTGTT 45 preCoreHBPr46 TGTTCATGTCCTACTGTT 46 preCore HBPr47 ACTGTTCAAGCCTCCAAG 47preCore HBPr48 GGCACAGGCTTGGAGGCTT 48 preCore HBPr49 AAAGCCACCCAAGGCACA49 preCore HBPr50 CCCAGAGGGTTGGGAAC 50 preS1 HBPr51 CAGCATGGGGCAGAATCT51 preS1 HBPr52 TCCACCAGCAATCCTCTG 52 preS1 HBPr53 GGATCCAGCCTTCAGAGC 53preS1 HBPr54 TCAGGAAGACAGCCTAC 54 preS1 HBPr55 TTCAACCCCAACAAGGATC 55preS1 HBPr56 AATGCTCCAGCTCCTAC 56 preS1 HBPr57 CTGCATTCAAAGCCAACT 57preS1 HBPr58 CCCCATGGGGGACTGTTG 58 preS1 HBPr59 CATACTCACAACTGTGCCA 59preS1 HBPr60 GGGCTTTCTTGGACGGTCC 60 preS1 HBPr61 CTCTCGAATGGGGGAAGA 61preS1 HBPr62 CCTACCCCAATCACTCCA 62 preS1 HBPr63 AGCACCTCTCTCAACGACA 63preS1 HBPr64 GCAAATTCCAGCAGTCCCG 64 preS1 HBPr65 GCCAATGGCAAACAAGGTA 65preS1 HBPr66 GACATGAACATGAGATG 66 preCore HBPr67 GGACATGAACAAGAGAT 67preCore HBPr68 GACATGTACAAGAGATG 68 preCore HBPr69 ACATAAGAGGACTCTTGGAC69 preCore primer sense HBPr70 TACTTCAAAGACTGTGTGTTTA 70 preCore primersense HBPr71 ACAAAGACCTTTAAC/TCT 71 preCore promoter HBPr72ACAAAGATCATTAAC/TCT 72 preCore promoter HBPr73 TTCCACCAGCAATCCTC 73preS1 HBPr74 GATCCAGCCTTCAGAGC 74 preS1 HBPr75 CAAGGTATGTTGCCCGTTTGTCC75 HBsAg primer sense HBPr76 CCAAACAGTGGGGGAAAGCCC 76 HBsAg primeranti-sense HBPr77 CTACGGATGGAAATTGC 77 HBsAg codon 145 wild type HBPr78TACGGACGGAAACTGC 78 HBsAg codon 145 wild type HBPr79 TTCGGACGGAAACTGC 79HBsAg codon 145 wild type HBPr80 CTTCGGACGGAAATTGC 80 HBsAg codon 145wild type HBPr81 CTACGGATAGAAATTGC 81 HBsAg codon 145 mutant HBPr82CTTCGGACAGAAATTGC 82 HBsAg codon 145 mutant HBPr83CTATGGGAGTGGGCCTCAGT/CC 83 HB Pol HBPr84 GCTGTAGGCATAAATTGGTCTG 84preCore primer sense HBPr85 CTCCACAGT/AAGCTCCAAATTC 85 preCore primeranti-sense HBPr86 ACATAAGAGGACTCTTGGAC 86 preCore primer sense HBPr87TACTTCAAAGACTGTGTGTTTA 87 preCore primer sense HBPr88 TAGGTTAAAGGTCTTTGT88 preCore promoter HBPr89 TAGGTTAATGATCTTTGT 89 preCore promoter HBPr90CATGTCCCACTGTTTAA 90 preCore HBPr91 CATGTCCTACTGTTCAA 91 preCore HBPr92TTCTGCCCCATGCTGTA 92 preS1 HBPr93 TTCTGCCCCATGCTGTAG 93 preS1 HBPr94GGTAA/TAAAGGGACTCAC/AGATG 94 HBsAg primer anti-sense HBPr95TCAGCTATATGGATGAT 95 HB Pol HBPr96 CAGCTATATGGATGAT 96 HB Pol HBPr97TTCAGCTATATGGATG 97 HB Pol HBPr98 TCAGTTATATGGATGAT 98 HB Pol HBPr99TTTCAGTTATATGGATG 99 HB Pol HBPr100 TTTAGTTATATGGATGA 100 HB Pol HBPr101TCAGCTATGTGGATGAT 101 HB Pol HBPr102 TCAGTTATGTGGATGAT 102 HB PolHBPr103 TTTCAGCTATGTGGATG 103 HB Pol HBPr104 CAAGGTATGTTGCCCGTTTGTCC 104HBsAg primer sense HBPr105 GGT/CAA/TAAAGGGACTCAC/AGATG 105 HBsAg primeranti-sense HBPr106 GGGTCACCATATTCTTGGG 106 preS1 primer sense HBPr107GTTCCT/GGAACTGGAGCCACCAG 107 preS1 primer anti-sense HBPr108CCGGAAAGCTTGAGCTCTTCTTTTTCACCTCTGCCTAATC 108 preCore primer senseHBPr109 CCGGAAAGCTTGAGCTCTTCAAAAAGTTGCATGGTGCTGG 109 preCore primeranti-sense HBPr110 CCTCTGCCGATCCATACTGCGGAAC 110 preX primer senseHBPr111 CTGCGAGGCGAGGGAGTTCTTCTTC 111 HB Core primer anti-sense HBPr112TGCCATTTGTTCAGTGGTTCGTAGGGC 112 HBsAg primer sense HBPr113CCGGCAGATGAGAAGGCACAGACGG 113 HBX primer antisense HBPr114TTCAGCTATATGGATGAT 114 YMDD motif HBPr115 TCAGCTATATGGATGATG 115 YMDDmotif HBPr116 TTCAGCTATGTGGATGAT 116 YMDD motif HBPr117TCAGCTATGTGGATGATG 117 YMDD motif HBPr118 GGCTTTGGGGCATGG 118 preCorecodon 28 wild type HBPr119 TGGCTTTGGGGCATG 119 preCore codon 28 wildtype HBPr120 GTGGCTTTGGGGCATG 120 preCore codon 28 wild type HBPr121GGCTTTGGGGCATGGA 121 preCore codon 28 wild type HBPr122 TGGCTTTGGGACATGG122 preCore codon 28 wild type, codon 29 mutant HBPr123 GGCTTTGGGACATGG123 preCore codon 28 wild type, codon 29 mutant HBPr124 TGGCTTTGGGACATG124 preCore codon 28 wild type, codon 29 mutant HBPr125 GTGGCTTTGGGACATG125 preCore codon 28 wild type, codon 29 mutant HBPr126 GGCTTTGGGACATGGA126 preCore codon 28 wild type, codon 29 mutant HBPr127TCAGTTATATGGATGATG 127 YMDD genotype D, wild type HBPr128TTCAGTTATATGGATGAT 128 YMDD genotype D, wild type HBPr129TTTCAGTTATATGGATGAT 129 YMDD genotype D, wild type HBPr130TCAGTTATGTGGATGATG 130 YMDD genotype D, mutant HBPr131TTCAGTTATGTGGATGAT 131 YMDD genotype D, mutant HBPr132TTTCAGTTATGTGGATGAT 132 YMDD genotype D, mutant HBPr133TTTCAGTTATGTGGATGA 133 YMDD genotype D, mutant HBPr134TGCTGCTATGCCTCATCTTC 134 outer HBsAg primer sense HBPr135CA(G/A)AGACAAAAGAAAATTGG 135 outer HBsAg primer anti-sense HBPr136CTATGGATGGAAATTGC 136 HBsAg mutant codon 143 HBPr137 CCTATGGATGGAAATTG137 HBsAg mutant codon 143 HBPr138 ACCTATGGATGGAAATT 138 HBsAg mutantcodon 143 HBPr139 CT CAA GGC AAC TCT ATG TGG 139 HBsAg, genotype AHBPr140 CT CAA GGC AAC TCT ATG GG 140 HBsAg, genotype A HBPr141 T CAAGGC AAC TCT ATG TTG 141 HBsAg, genotype A HBPr142 ATC CCA TCA TCT TGG G142 HBsAg, genotype B HBPr143 ATC CCA TCA TCT TGG GCG G 143 HBsAg,genotype B HBPr144 TC CCA TCA TCT TGG GCG G 144 HBsAg, genotype BHBPr145 C CCA TCA TCT TGG GCT GG 145 HBsAg, genotype B HBPr146 TTC GCAAAA TAC CTA TGG 146 HBsAg, genotype B HBPr147 T TTC GCA AAA TAC CTA TG147 HBsAg, genotype B HBPr148 CT TTC GCA AAA TAC CTA TG 148 HBsAg,genotype B HBPr149 TC GCA AAA TAC CTA TGG G 149 HBsAg, genotype BHBPr150 T CTA CTT CCA GGA ACA T 150 HBsAg, genotype C HBPr151 T CTA CTTCCA GGA ACA TC 151 HBsAg, genotype C HBPr152 CT CTA CTT CCA GGA ACA T152 HBsAg, genotype C HBPr153 CT CTA CTT CCA GGA ACA G 153 HBsAg,genotype C HBPr154 C TGC ACG ATT CCT GCT 154 HBsAg, genotype C HBPr155TGC ACG ATT CCT GCT CA 155 HBsAg, genotype C HBPr156 C TGC ACG ATT CCTGCT C 156 HBsAg, genotype C HBPr157 TGC ACG ATT CCT GCT CAA 157 HBsAg,genotype C HBPr158 TTC GCA AGA TTC CTA TG 158 HBsAg, genotype C HBPr159CT TTC GCA AGA TTC CTA T 159 HBsAg, genotype C HBPr160 CT TTC GCA AGATTC CTA 160 HBsAg, genotype C HBPr161 CT TTC GCA AGA TTC CTA TG 161HBsAg, genotype C HBPr162 C TCT ATG TAT CCC TCC T 162 HBsAg, genotype DHBPr163 TCT ATG TAT CCC TCC TG 163 HBsAg, genotype D HBPr164 C TCT ATGTAT CCC TCC TGG 164 HBsAg, genotype D HBPr165 CC TCT ATG TAT CCC TCC T165 HBsAg, genotype D HBPr166 C TGT ACC AAA CCT TCG G 166 HBsAg,genotype D HBPr167 C TGT ACC AAA CCT TCG 167 HBsAg, genotype D HBPr168GC TGT ACC AAA CCT TCG G 168 HBsAg, genotype D HBPr169 TGT ACC AAA CCTTCG GAG 169 HBsAg, genotype D HBPr170 GGA CCC TGC CGA ACC T 170 HBsAg,genotype E HBPr171 GGA CCC TGC CGA ACC G 171 HBsAg, genotype E HBPr172 GGGA CCC TGC CGA AC 172 HBsAg, genotype E HBPr173 GGA CCC TGC CGA AC 173HBsAg, genotype E HBPr174 GT TGC TGT TCA AAA CCT T 174 HBsAg, genotype EHBPr175 GT TGC TGT TCA AAA CCT G 175 HBsAg, genotype E HBPr176 TGT TGCTGT TCA AAA CCT G 176 HBsAg, genotype E HBPr177 A TGT TGC TGT TCA AAACCT G 177 HBsAg, genotype E HBPr178 GA TCC ACG ACC ACC A 178 HBsAg,genotype F HBPr179 GGA TCC ACG ACC ACC A 179 HBsAg, genotype F HBPr180GGA TCC ACG ACC ACC 180 HBsAg, genotype F HBPr181 GA TCC ACG ACC ACC AGG181 HBsAg, genotype F HBPr182 TGT TCC AAA CCC TCG G 182 HBsAg, genotypeF HBPr183 C TGT TCC AAA CCC TCG 183 HBsAg, genotype F HBPr184 C TGT TCCAAA CCC TCG G 184 HBsAg, genotype F HBPr185 GT TCC AAA CCC TCG GAT 185HBsAg, genotype F HBPr186 G CCA AAT CTG TGC AGC 186 HBsAg, genotype FHBPr187 CCA AAT CTG TGC AGC AT 187 HBsAg, genotype F HBPr188 G CCA AATCTG TGC AGC AG 188 HBsAg, genotype F HBPr189 GG CCA AAT CTG TGC AGC 189HBsAg, genotype F HBPr190 A TCA ACA ACA ACC AGT A 190 HBsAg, genotype AHBPr191 GA TCA ACA ACA ACC AGT 191 HBsAg, genotype A HBPr192 GA TCA ACAACA ACC AGT A 192 HBsAg, genotype A HBPr193 GGA TCA ACA ACA ACC AGT 193HBsAg, genotype A HBPr194 T CAA GGC AAC TCT ATG TGG 194 HBsAg, genotypeA HBPr195 AGG TTA AAG GTC TTT GT 195 promoter genotype A wild typeHBPr196 T AGG TTA AAG GTC TTT GG 196 promoter genotype A wild typeHBPr197 TT AGG TTA AAG GTC TTT 197 promoter genotype A wild type HBPr198GG TTA AAG GTC TTT GTA GG 198 promoter genotype A wild type HBPr199 AGGTTA ATG ATC TTT GT 199 promoter genotype A mutant HBPr200 T AGG TTA ATGATC TTT GG 200 promoter genotype A mutant HBPr201 CT TTC GCA AGA TTC CTATGG 201 HBsAg genotype C codon 160 HBPr202 GCT TTC GCA AGA TTC CTA TG202 HBsAg genotype C codon 160 HBPr203 GCT TTC GCA AGA TTC CTA TGG 203HBsAg genotype C codon 160 HBPr204 CT TTC GCA AGA TTC CTA TGG G 204HBsAg genotype C codon 160 HBPr205 GC TGT ACC AAA CCT TCG GAG 205 HBsAggenotype D codon 140 HBPr206 TGC TGT ACC AAA CCT TCG G 206 HBsAggenotype D codon 140 HBPr207 TGC TGT ACC AAA CCT TCG GAG 207 HBsAggenotype D codon 140 HBPr208 GC TGT ACC AAA CCT TCG GAT 208 HBsAggenotype D codon 140 HBPr209 TGG TTC GCC GGG CTT T 209 HBsAg genotype Ecodon 184 HBPr210 G TGG TTC GCC GGG CTT G 210 HBsAg genotype E codon 184HBPr211 GG TTC GCC GGG CTT TC 211 HBsAg genotype E codon 184 HBPr212 TGGTTC GCC GGG CTT TC 212 HBsAg genotype E codon 184 HBPr213 AG TGG TTC GCCGGG CTG G 213 HBsAg genotype E codon 184 HBPr214 A GGA TCC ACG ACC ACCAGG 214 HBsAg genotype F HBPr215 A GGA TCC ACG ACC ACC AGT 215 HBsAggenotype F HBPr216 CA GGA TCC ACG ACC ACC AGG 216 HBsAg genotype FHBPr217 C TGT TCC AAA CCC TCG GAG 217 HBsAg genotype F HBPr218 C TGT TCCAAA CCC TCG GAT 218 HBsAg genotype F HBPr219 GC TGT TCC AAA CCC TCG GAG219 HBsAg genotype F HBPr220 CTGAACCTTTACCCCGTTGC 220 enhancer primerHBPr221 CTCGCCAACTTACAAGGCCTTTC 221 enhancer primer HBPr222AGAATGGCTTGCCTGAGTGC 222 Core primer anti-sense HBPr223 GCT TTC GCA AGATTC CTA TGG G 223 HBsAg genotype C codon 160 HBPr224 G GCT TTC GCA AGATTC CTA TGG 224 HBsAg genotype C codon 160 HBPr225 G GCT TTC GCA AGA TTCCTA TGG G 225 HBsAg genotype C codon 160 HBPr226 G GCT TTC GCA AGA TTCCTA TGG GA 226 HBsAg genotype C codon 160 HBPr227 C AGC TAT ATG GAT GATGTG 227 YMDDV motif HBPr228 AGC TAT ATG GAT GAT GTG GG 228 YMDDV motifHBPr229 GC TAT ATG GAT GAT GTG GT 229 YMDDV motif HBPr230 AGC TAT ATGGAT GAT GTG GT 230 YMDDV motif HBPr231 C AGC TAT ATG GAT GAT ATA 231YMDDI MOTIF HBPr232 AGC TAT ATG GAT GAT ATA GG 232 YMDDI MOTIF HBPr233GC TAT ATG GAT GAT ATA GT 233 YMDDI MOTIF HBPr234 AGC TAT ATG GAT GATATA GT 234 YMDDI MOTIF HBPr235 CCA TCA TCT TGG GCT TG 235 HBSAg GENOTYPEB CODON 155 HBPr236 CA TCA TCT TGG GCT TT 236 HBSAg GENOTYPE B CODON 155HBPr237 CCA TCA TCT TGG GCT TT 237 HBSAg GENOTYPE B CODON 155 HBPr238CCA TCA TCT TGG GCT TTC 238 HBSAg GENOTYPE B CODON 155 HBPr239 CCC ACTGTC TGG CTT TC 239 HBSAg GENOTYPE B CODON 190 HBPr240 CC ACT GTC TGG CTTTC 240 HBSAg GENOTYPE B CODON 190 HBPr241 CC ACT GTC TGG CTT T 241 HBSAgGENOTYPE B CODON 190 HBPr242 CCC ACT GTC TGG CTT G 242 HBSAg GENOTYPE BCODON 190 HBPr243 TAT ATG GAT GAT GTG GTA 243 YMDDV MOTIF HBPr244 TATGTG GAT GAT GTG GTA 244 YVDDV MOTIF HBPr245 TAT ATA GAT GAT GTG GTA 245YIDDV MOTIF HBPr246 TAT ATT GAT GAT GTG GTA 246 YIDDV MOTIF HBPr247 TATGTA GAT GAT GTG GTA 247 YVDDV MOTIF HBPr248 TAT GTT GAT GAT GTG GTA 248YVDDV MOTIF HBPr249 TAT ATG GAT GAT ATA GTA 249 YMDDI MOTIF HBPr250 TATATG GAT GAT ATC GTA 250 YMDDI MOTIF HBPr251 TAT GTG GAT GAT ATA GTA 251YVDDI MOTIF HBPr252 TAT GTG GAT GAT ATC GTA 252 YVDDI MOTIF HBPr253 TATATA GAT GAT ATA GTA 253 YIDDI MOTIF HBPr254 TAT ATA GAT GAT ATC GTA 254YIDDI MOTIF HBPr255 TAT ATT GAT GAT ATA GTA 255 YIDDI MOTIF HBPr256 TATATT GAT GAT ATC GTA 256 YIDDI MOTIF HBPr257 TAT GTA GAT GAT ATA GTA 257YVDDI MOTIF HBPr258 TAT GTA GAT GAT ATC GTA 258 YVDDI MOTIF HBPr259 TATGTT GAT GAT ATA GTA 259 YVDDI MOTIF HBPr260 TAT GTT GAT GAT ATC GTA 260YVDDI MOTIF HBPr261 TAT ATG GAT GAT CTG GTA 261 YMDDL MOTIF HBPr262 TATGTG GAT GAT CTG GTA 262 YVDDL MOTIF HBPr263 TAT ATA GAT GAT CTG GTA 263YIDDL MOTIF HBPr264 TAT ATT GAT GAT CTG GTA 264 YIDDL MOTIF HBPr265 TATGTA GAT GAT CTG GTA 265 YVDDL MOTIF HBPr266 TAT GTT GAT GAT CTG GTA 266YVDDL MOTIF HBPr267 T ATG GGA GTG GGC CTC AG 267 MGVGL HBPr268 T ATG GGATTG GGC CTC AG 268 MGLGL HBPr269 C AGT CCG TTT CTC TTG GC 269 SPFLL

EXAMPLES Example 1 HBV DNA Preparation and PCR Amplification

Serum samples were collected from HBsAg-positive individuals and storedat minus 20° C. until use in 0.5 ml aliquots. To prepare the viralgenome, 18 μl serum was mixed with 2 μl 1N NaOH and incubated at 37° C.for 60 minutes. The denaturation was stopped and neutralized by adding20 μl of 0.1N HCl. After a 15 minutes centrifugation step, thesupernatant was collected and the pellet discarded. PCR was carried outon this lysate as follows: 32 μl H₂O was mixed with 5 μl of 10× PCRbuffer, 1 μl 10 mM dXTPs, 1 μl of each biotinylated primer (10 pmol/μl),10 μl of serum lysate, and 2 U Taq enzyme. The amplification schemecontained 40 cycles of 95° C. 1 min, annealing at 45° C. for 1 min, andextension at 72° C. for 1 min. Amplification products were visualized on3% agarose, gel.The outer primer set for preS1 has the following sequence:

outer sense: HBPr 1: 5′-bio-GGGTCACCATATTCTTGGG-3′ outer antisense HBPr4: 5′-bio-GTTCC(T/G)GAACTGGAGCCACCAG-3′ The outer primer set for preCorehas the following sequence: outer sense: HBPr 69:5′-bio-ACATAAGAGGACTCTTGGAC-3′ outer antisense: HBPr 8:5′-bio-GAAGGAAAGAAGTCAGAAGGC-3′ The outer primer set for HBAg has thefollowing sequence: outer sense: HBPr 134:5′-bio-TGCTGCTATGCCTCATCTTC-3′ outer antisense: HBPr 135:5′-bio-CA(G/A)AGACAAAAGAAAATTGG˜3′.Samples that were negative in the first round PCR were retested in anested reaction composed of the following: μl H₂O, 5 μl 10× Taq buffer,1 μl 10 mM dXTPs, 1 μl of each nested primer (10 pmol/μl), 1 μl of thefirst round PCR product, and 2 U Taq polymerase. The amplificationscheme was identical as for the first round PCR. The sequence of thenested primers were as follows, for the preS1 region:

nested sense HBPr 2: 5′-bio-GAACAAGAGCTACAGCATGGG-3′ nested antisenseHBPr 3: 5′-bio-CCACTGCATGGCCTGAGGATG-3′; and for the preCore region:nested sense HBPr 70: 5′-bio-TACTTCAAAGACTGTGTGTTTA-3′ nested antisenseHBPr 7: 5′-bio-CTCCACAG(T/A)AGCTCCAAATTC-3′ In a second reaction theHBsAg region can be amplified in a similar protocol by using thefollowing primers: HBPr 75: 5′-bio-CAAGGTATGTTGCCCGTTTGTCC-3′ incombination with either HBPr 76: 5′-bio-CCAAACAGTGGGGGAAAGCCC-3′; orwith HBPr 94: 5′-bio-GGTA(A/T)AAAGGGACTCA(C/A)GATG-3′.

Example 2 Preparation of the Line Probe Assays

Probes were designed to cover the universal, genotypic and mutantmotifs, in principle only probes that discriminate between one singlenucleotide variation were retained. However, for certain polymorphismsat the extreme ends of the probe, cross-reactivity was tolerated.Specificity was reached experimentally for each probe individually afterconsidering the % (G+C), the probe length, the final concentration, andhybridization temperature. Optimized probes were provided enzymaticallywith a poly-T-tail using the TdT (Pharmacia) in a standard reactioncondition. Briefly, 400 pmol probe was incubated at 37° C. in a 30 μlreaction mix containing 5.3 mM dTTP, 25 mM Tris.HCL pH 7.5, 0.1 M sodiumcacodylate, 1 mM CoCl₂, 0.1 M DTT and 170 U terminal deoxynucleotidyltransferase (Pharmacia). After one hour incubation, the reaction wasstopped and the tailed probes were precipitated and washed with ice-coldethanol. Probes were dissolved in 6× SSC at their respectively specificconcentrations and applied as horizontal lines on membrane strips inconcentrations between 0.2 and 2.5 pM/ml. Biotinylated DNA was appliedalongside as positive control (LiPA line 1). The oligonucleotides werefixed to the membrane by baking at 80° C. for 12 hours. The membrane wasthan sliced into 4 mm strips. The design of this strip is indicated inFIG. 2.

Example 3 LiPA Test Performance

Equal volumes (10 μl each) of the biotinylated PCR fragment and of thedenaturation solution (DS; 400 mM NaOH/10 mM EDTA) were mixed in testtroughs and incubated at room temperature for 5 minutes. Then, 2 ml ofthe 37° C. prewarmed hybridization solution (HS, 3× SSC/0.1% SDS) wasadded, followed by the addition of one strip per test trough.Hybridisation occured for 1 hour at 50±0.5° C. in a closed shaking waterbath. The strips were washed twice with 2 ml of stringent wash solution(3× SSC/0.1% SDS) at room temperature for 20 seconds, and once at 50° C.for 30 minutes. Following this stringent wash, strips were rinsed twotimes with 2 ml of the Innogenetics standard Rinse Solution (RS). Stripswere incubated on a rotating platform with the alkalinephosphatase-labelled streptavidin conjugate, diluted in standardConjugate Solution for 30 minutes at room temperature (20 to 25° C.).Strips were than washed twice with 2 ml of RS and once with standardSubstrate Buffer (SB), and the colour reaction was started by addingBCIP and NBT to the SB. After maximum 30 minutes at room temperature,the colour reaction was stopped by replacing the colour compounds bydistilled water. Immediately after drying, the strips were interpreted.Reactivities were considered positive whenever the reactivity wasstronger than the reaction on the negative control. Strips can be storedon a dry dark place. The complete procedure described above can also bereplaced by the standardized Inno-LiPA automation device (auto-LiPA).

Example 4 Selection of Reference Material

PCR fragments were prepared, derived from members of the differentgenotypes, the different preCore wild type and mutant sequences, drugresistant motifs and vaccine escape mutants. The PCR fragments wereamplified with primers lacking the biotine group at their 5′-end andcloned into the pretreated EcoRV site of the pGEMT vector (Promega).Recombinant clones were selected after α-complementation and restrictionfragment length analysis, and sequenced with plasmid primers. Otherbiotinylated fragments were directly sequenced with a dye-terminatorprotocol (Applied Biosystems) using the amplification primers.Alternatively, nested PCR was carried out with analogs of the primers,in which the biotine group was replaced with the T7- and SP6-primersequence, respectively. These amplicons were than sequenced with an SP6-and T7-dye-primer procedure. By doing so, a reference panel ofrecombinant clones was prepared, which is necessary for optimizing LiPAprobes.

Example 5 Genotyping HBV-Infected Serum Samples

Only after creating a sequence alignment as shown in FIG. 1, it becameclear which regions could be useful for HBV genotyping. The preS1 regionseems to be suitable because of the high degree of variability. Probeswere therefore designed to cover most of these variable regions as shownin Table 1. Only a limited selection of probes was retained because oftheir specific reaction with the reference panel. The most importantones are indicated as boxed regions in FIG. 1. These selected probeswere then applied in a LiPA format indicated in FIG. 2, as line number 2to 14. Some of the probes could be applied together in one line, becauseof their universal character, while others need to be appliedseparately. With the selection of probes thus obtained, serum samplescollected in different parts of the world (Europe, South-America,Africa, Middle-East) were tested. The upper part of FIG. 3 shows thereactivity of a selection of samples on these probes. Genotyping ofthese samples is straightforward, with samples 2 to 8 belonging togenotype A, samples 9 and 10 belonging to genotype B, samples 11 and 12belonging to genotype C, samples 13 to 19 belonging to genotype D,samples 20 to 23 belonging to genotype E, and sample 24 belonging togenotype F.

Genotyping can also be performed in the HBsAg region. Again, probes weredesigned to cover most of the variable, regions shown in FIG. 1. Only alimited selection of probes were retained. These probes are boxed inFIG. 1 and are listed in FIG. 4. A LiPA strip was prepared carryingthese probes and samples belonging to the different genotypes werecharacterized, as shown in FIG. 5.

Example 6 Scanning the PreCore Region for Mutations

HBeAg expression can be regulated at the transcriptional andtranslational level. It is postulated that a transcriptional regulationexists due to the presence of a dinucleotide variation in the promoterregion of the preCore mRNA. Probes covering the wild type (e.g. probeHBPr 88) and the mutant (e.g. HBPr 89) motif were selected and theirpositions are indicated in the alignment shown in FIG. 1, and applied onthe LiPA strip as line 15 and 16 (FIG. 2).

At the translational level, much more mutations might arise, allpossibly resulting in abrogation of the HBeAg expression: any mutationsat codon 1 (ATG) destroying translation initiation, codon 2 (CAA toTAA), codon 7 (TGC to TGA), codon 12 (TGT to TGA), codon 13 in genotypeB, C, D, E, F (TCA to TGA or TAA), codon 14 (TGT to TGA), codon 18 (CAAto TAA), codon 21 (AAG to TAG), codon 23 (TGC to TGA), codon 26 (TGG toTAG or TGA), codon 28 (TGG to TAG or TGA). However, due to secondarycontrain of the encapsidation signal, most of the mutations occur atcodon 28 (TGG to TAG). Along with the mutation at codon 28, a secondmutation at codon 29 (GGC to GAC) is often observed. In the case ofgenotype A and again as a consequence of the secondary constrain, stopcodon mutations at codon 28 are only likely to occur after selection ofa codon 15 mutation (CCC to CCT). Hence, correct interpretation ofpreCore mutations is genotype dependent. In addition to the abovementioned stop codons, a huge amount of different deletion- orinsertion-mutations in the preCore open reading frame might giveessentially the same result.

In order to develop a sensitive assay to detect the relevant mutationsand the hypothetical mutations, a probe scanning procedure wasdeveloped. Partially overlapping probes were designed and applied in aLiPA format (FIG. 2, line 17 to 27). In this assay format, wild typesequences over the complete preCore region, together with the codon 15variation for genotype A versus non-A genotypes, and the most commonmutations at codon 28 (TAG), at codon 29 (GAC) and the combination ofcodon 23 and 29 (TAGGAC) are positively recognized. Absence ofreactivity at one of the other probes is always indicative for thepresence of a variation. The exact nature of this variation can then berevealed by sequence analysis or with further designed LiPA probes.

FIG. 3 shows the reactivity of the selected genotyped samples on theprobes for the preCore region. Samples were previously tested for thepresence of HBeAg or for anti-HBe. The interpretation of the reactivityon the LiPA probes for each sample is indicated below each strip. Thisapproach allowed for the simultaneous screening of a sample for preCoremutations and the characterization of the viral genotype.

FIG. 6 also shows a panel of samples with mutations in the preCoreregion, as well as wild type samples. The probes used in this assay arelisted in FIG. 4. This assay includes a codon 29 mutant (M4 motif),which was not present in the experiment in FIG. 3.

Example 7 Detection of Mutants in the HBsAg Region

Vaccine escape mutants have been described. The most commonly foundmutant is the variation at codon 145 of HBsAg (G145R or GGA to AGA).LiPA probes are designed to detect wild type and mutant probes.Genotypic variations are present in the vicinity of codon 145.Therefore, genotype A is covered by probe 77, genotype B by probe 78,genotype C by probe 79, and genotype D/E by probe 80. Hence, inprinciple, it is possible to genotype and detect the wild type strainsof the virus in one single experiment. Mutant target sequences arecovered by probe 81 and 82 for genotype A and D, respectively. Probe 83can be used as a positive control in these experiments. Furtherdetection of mutants in the a determinant region is possible by means ofa probe scanning approach. Herefore, probes are designed to cover thewild type sequence of the different genotypes over the HBsAg epitoperegion and applied in a LiPA format. Again here, absence of staining axone of these probes is indicative for the presence of a mutant strain.The exact nature of this variant is then determined by sequencinganalysis.

Example 8 Detection of HBV Strains Resistant to Lamivudine

Through analogy with HIV and the resistance against the anti-viralcompound 3TC (lamivudine or (−)-β-1-2′,3′-dideoxy-3′-thiacytidine), itwas predicted that upon treatment of HBV-infected patients with 3TC,viral strains would be selected showing resistance at the YMDD motif inthe HB pol gene. The YMDD motif is physically located in the HBsAgregion, but is encoded in another reading frame. Hence, this part of theHBV pol region is amplified with the primer combination HBPr 74-HBr 94,but not with the combination HBPr 74-HBr 76. Probes covering the wildtype YMDD motif and YVDD mutant motif are indicated in FIG. 1,respectively probes 95 to 100 and 101 to 103, as well as probes 115,116, 127 and 132, the latter probes yielding the best results in theLiPA assay. Such an assay was used to determine the presence ofmutations in the YMDD motif in serum of a HBV-infected patient duringtreatment with lamivudine. FIG. 7 shows that in the first phase of thetreatment (May 1995) no mutations were detected. During the treatment,the viral load decreased, reaching a level of approximately 10⁴ duringNovember and December 1995, whereafter a breakthrough was observed,resulting in a level as high as during the first months of the treatmentby June 1996. Interestingly, a LiPA assay performed in February 1996indicated that the majority of virus present, possessed a mutation inthe YMDD motif, which had changed to YVDD. In June 1996, no more wildtype motif, but only mutant YVDD could be detected. With this assay,resistant HBV strains can thus easily be detected. Furthermore, thecombined detection of the YMDD motif and preCore mutants might beclinically important in prediction and prognosis of further treatment.

References

-   Asseline U, Delarue M, Lancelot G, Toulme F, Thuong N (1984) Nucleic    acid-binding molecules with high affinity and base sequence    specificity: intercalating agents covalently linked to    oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 81(11):3297-301.-   Barany F. Genetic disease detection and DNA amplification using    cloned thermostable ligase. Proc Natl Acad Sci USA 1991; 88:    189-193.-   Bej A, Mahbubani M, Miller R, Di Cesare J, Haff L, Atlas R. Mutiplex    PCR amplification and immobilized capture probes for detection of    bacterial pathogens and indicators in water. Mol Cell Probes 1990;    4: 353-365.-   Boom R., Sol C. J. A., Salimans M. M. M., et al. Rapid and simple    method for purification of nucleic acids. J Clin Microbiol 1990; 28:    495-503.-   Carman W, Zanetti A, Karayiannis P, Waters J, Manzillo G, Tanzi E,    Zuckerman A, and Thomas H. Vaccine induced escape mutants. Lancet    1990; 336: 325-329.-   Carman W, Koruia J, Wallace L, MacPhee R, Mimms L, and Decker R.    Fulminant reactivation of hepatitis B due to envelope protein mutant    that escaped detection by monoclonal HBsAG ELISA. Lancet 1995; 345:    1405-1407.-   Compton J. Nucleic acid sequence-based amplification. Nature 1991;    350: 31-92.-   Crawford D. hepatitis B virus ‘escape’ mutants: A rare event which    causes vaccinationfailare. British Med. J. 1990; 301: 1058-1059.-   Duck P. Probe amplifier system based on chimeric cycling    oligonucleotides. Biotechniques 1990; 9: 142-147.-   Gao Q, Gu Z, Parniak M, Cameron I, Cammack N, Boucher C, and    Wainberg M. The same mutation that encodes low-level human    immunodeficiency virus type-1 resistance to 2′,3′-dideoxyinosine and    2′,3′-dideoxycytidine confers high level resistance to the (−)    enantiomer of 2′,3′-dideoxy-3′-thiacytidine. Antimicrob. Agents    Chemother. 1993; 37: 1390-1392.-   Guatelli J, Whitfield K, Kwoh D, Barringer K, Richman D, Gengeras T.    Isothermal, in vitro amplification of nucleic acids by a multienzyme    reaction modeled after retroviral replication. Proc Natl Acad Sci    USA 1990; 87: 1874-1878.-   Hadziyannis S. Hepaptis B e antigen negative chronic hepatitis B:    from clinical recognition to pathogenesis and treatment. Viral    Hepatitis 1995; 1: 7-36.-   Honkoop P, de Man R, Zondervan P, Niesters H, and Schalm S.    Histological improvement in patients with chronic hepatitis B virus    infection treated with lavimudine is associated with a decrease in    HBV-DNA by PCR. Hepatol. 1995; 22: abstract 887.-   Kwoh D, Davis G, Whitfield K, Chappelle H, Dimichele L, Gingeras T.    Transcription-based amplification system and detection of amplified    human immunodeficiency virus type 1 with a bead-based sandwich    hybridization format. Proc Natl Acad Sci USA 1989; 86: 1173-1177.-   Kwok S, Kellogg D, McKinney N, Spasic D, Gods L, Levenson C,    Sinisky J. Effects of primer-template mismatches on the polymerase    chain reaction: Human immunodeficiency views type 1 model studies.    Nucl. Acids Res. 1990; 18:999.-   Landgren U, Kaiser R, Sanders J, Hood L. A ligase-mediated gene    detection technique. Science 1988; 241: 1077-1080.-   Li J-S, Tong S-P, Wen Y-M, Vivitski L, Zhang Q, and Trépo C.    Hepatitis B virus genotype A rarely circulates as an Hbe-minus    mutant: possible contribution of a single nucleotide in the preCore    region. J. Virol. 1993; 67: 5402-5410.-   Ling, R., Mutimer, D., Ahmed, M., Boxall, E. H., Elias, E.,    Dusheiko, G. M. and Harrison, T. J. Selection of mutations in the    Hepatitis B Virus polymerase during therapy of transplant recipients    with lamivudune. Hepatology 1996; 24: 711-713.-   Lok A, Akarca U, and Greene S. Mutations in the precore region of    hepatitis B virus serve to enhance of the secondary structure of the    pre-genome encapsidation signal. Proc. Natl. Acad. Sci. USA 1994;    91: 4077-4081.-   Lomeli H, Tyagi S, Printchard C, Lisardi P, Kramer F. Quantitative    assays based on the use of replicatable hybridization probes. Clin    Chem 1989; 35: 1826-1831.-   Magnius L, and Norder H. Subtypes, genotypes and molecular    epidemiology of the hepatitis B virus as reflected by sequence    variability of the S-gene. Intervirology 1995; 38: 24-34.-   Matsukura M, Shinozuka K, Zon G, Mitsuya H, Reitz M, Cohen J, Broder    S (1987) Phosphorothioate analogs of oligodeoxynucleotides:    inhibitors of replication and cytopathic effects of human    immunodeficiency virus. Proc. Natl. Acad. Sci. USA 84(21):7706-10.-   Miller P, Yano J, Yano E. Carroll C, Jayaram K, Ts'o P (1979)    Nonionic nucleic acid analogues. Synthesis and characterization of    dideoxyribonucleoside methylphosphonates. Biochemistry    18(23):5134-43.-   Naoumov N, Perillo R, Chokshi S, Dienstag J, Vicary C, Brown N, and    Williams R. Reduction in hepatitis B virus quasispecies during    lamivudine treatment is associated with enhanced virus replication    and hepatocytolisis. Hepatol. 1995; 22: abstract 885.-   Nielsen P, Egholm M, Berg R, Buchardt O (1991) Sequence-selective    recognition of DNA by strand displacement with a thymine-substituted    polyamide. Science 254(5037):1497-500.-   Nielsen P, Egholm M, Berg R, Buchardt O (1993) Sequence specific    inhibition of DNA restriction enzyme cleavage by PNA.    Nucleic-Acids-Res. 21(2):197-200.-   Okamoto H, Yotsumoto S, Akahane Y, Yamanaka T, Miyazaki Y, Sugai Y,    Tsuda F, Tanaka T, Miyakawa Y, and Mayumi M. Hepatitis B virus with    precore region defects prevail in persistently infected hosts along    with seroconversion to the antibody against e antigen. J. Virol.    1990; 54: 1298-1303.-   Saiki R, Walsh P, Levenson C, Erlich H. Genetic analysis of    amplified DNA with immobilized sequence-specific oligonucleotide    probes Proc Natl Acad Sci USA 1989; 86: 6230-6234.-   Sato S, Suzuki K, Akahane Y, Akamatsu K, Akiyama K, Yunomura K,    Tsuda F, Tanaka T, Okamoto H, Miyakawa Y, Mayumi M. Hepatitis B    virus strains with mutations in the core promoter in patients with    fulminant hepatitis. Ann. Intern. Medicine 1995; 122: 241-248.-   Shaw, T., Mok, S. S., Locarnini, S.A. Inhibition of hepatitis B    virus DNA polymerase by enantiomers of penciclovir triphosphate and    metabolic basis for selective inhibition of HBV replication by    penciclovir. Hepatology 1996; 24:996-1002.-   Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virus    isolates and characterization of new subtypes using a iine probe    assay. J. Gen. Virol. 1993; 74: 1093-1102.-   Takahashi K, Aoyama K, Onhno N, Iwata K, Akahane Y, Baba K,    Yoshizawa H, and Mishiro S. The precore/core promoter mutant    (T1762A1764) of hepatitis B virus: clinical significance and an easy    method for detection. J. Gen. Virol. 1995; 76: 3159-3164.-   Tipples, G. A., Ma, M. M., Fischer, K. P., Bain, V. G.,    Kneteman, N. M. and Tyrell, D. L. J. Mutation in HBV RNA-dependent    DNA polymerase confers resistance to lamivudine in vivo. Hepatology    1996; 24: 714-717.-   Waters J, Kennedy M, Voet P, Hauser P, Petre J, Carman W, and    Thomas H. Loss of the common ‘a’ determinant of hepatitis B surface    antigen by vaccine-induced escape mutants. J. Clin. Invest. 1992;    90: 2543-2547.-   Wu D, Wallace B. The ligation amplification reaction    (LAR)—amplification of specific DNA sequences using sequential    rounds of template-dependent ligation. Genomics 1989; 4: 560-569.-   Yuan T, Faruqi A, Shih J, and Shih C. The mechanism of natural    occurrence of two closely linked HBV precore predominant mutations.    Virol. 1995; 211: 144-156.-   Zhang X, Zoulim F, Habersetzer F, Xiong S, and Trépo C. Analysis of    hepatitis B virus genotypes and preCore region variability during    interferon treatment of Hbe antigen negative chronic hepatitis B. J.    Med. Virol. 1996; xxx-xxx

The invention claimed is:
 1. A method for identifying a HBV strain resistant to lamivudine and penciclovir in a sample of serum of a HBV-infected patient, comprising: (i) amplifying at least a part of the HBV gene present in said sample with at least one primer pair; (ii) hybridizing the polynucleic acid products of the amplification of (i) with at least one probe which hybridizes specifically under stringent conditions to the codon coding for V of the YVDD motif of the HBV RT pol gene target sequence comprising the codon coding V of the YVDD motif; wherein said probe hybridizing specifically under stringent conditions to a HBV RT pol gene target sequence comprising the codon coding for the V of the YVDD motif is selected from the group consisting of SEQ ID NOs: 101-103, 116, 117, 130-133, 244-248, 251-260 and 262-266, or wherein said probe hybridizes specifically under stringent conditions to a complement of a sequence selected from the group consisting of 101-103, 116, 117, 130-133, 244-248, 251-260, and 262-266; (iii) based on the results of hybridizing (ii), determining the presence or absence of a mutation in the HBV RT pol gene resulting in an amino acid change in the YMDD motif of the HBV polymerase wherein said amino acid change is M to V in the YMDD motif and thereby identify the existence of a HBV strain resistant to lamivudine and penciclovir in said sample.
 2. A method according to claim 1 wherein one of the primers of the at least one primer pair is selected from the group consisting of SEQ ID NOs: 75, 94, 104, 105, 134 and
 135. 